1. Field of the Invention
This invention relates to methods and compositions for carrying out certain enzymatic reactions, specifically, to the recovery of nucleic acid from in vitro reaction mixtures that contain nucleases.
2. Description of the Related Art
Extensive study of the molecular biology of gene expression has led to improved methods for detection and quantitation of the messenger ribonucleic acid (mRNA) found in cells and tissues. A common method of mRNA analysis, called nuclease protection assays or ribonuclease protection assays, is performed routinely in many academic and medical research labs according to standard protocols. The standard protocols include obligatory steps for the recovery of undegraded nucleic acid from experimental reaction mixtures that contain degradative nuclease enzymes. Investigators will welcome further improvements that streamline the process of performing nuclease protection assays.
Analysis of gene expression often starts with the isolation of RNA from cells or tissues. Some of the requirements for isolation of RNA from biological sources are also requirements for successful analysis of the RNA using the method of nuclease protection assays. Specifically, both processes require recovery of intact nucleic acid from mixtures that contain nucleases (enzymes which degrade nucleic acid). Because the two processes are intertwined, the prior art will be reviewed as it relates to both RNA isolation from biological sources and RNA recovery from in vitro reactions. Fundamental differences between the two processes will then be considered.
Many currently used protocols for isolation of RNA from biological sources involve disruption of cells or tissue in concentrated solutions of guanidinium thiocyanate to inactivate endogenous nucleases and shear DNA, followed by phenol/chloroform extraction to denature and remove proteins. The RNA is recovered after the addition of ethanol or isopropanol which causes it to precipitate out of solution. (Chomczynski, et al., 1987) Sometimes recovery of RNA is accomplished by CsCl gradient centrifugation as an alternative to, or in addition to, alcohol precipitation. Other ingredients are often added to the guanidinium solution to aid in the inactivation of the nucleases. Examples include detergents (usually N-lauroyl sarcosine), reducing agents (e.g., dithiothreitol, 2-mercaptoethanol), and chelating agents (e.g., EDTA).
A typical procedure for isolation of RNA from mammalian tissue is found in Current Protocols in Molecular Biology (Ausbel, et al., 1987). This procedure involves homogenizing the tissue in a solution of guanidinium thiocyanate, Tris buffer, and .beta.-mercaptoethanol, centrifuging the suspension to remove particulate debris, adding N-lauroyl sarcosine, and sedimenting the RNA through a CsCl gradient by ultracentrifugation. The RNA pellet is resuspended in a solution of EDTA, N-lauroyl sarcosine, and .beta.-mercaptoethanol and then extracted with an equal volume of phenol:chloroform:isoamyl alcohol. After extraction, the solution is adjusted to 0.3M sodium acetate and the RNA is precipitated by the addition of 2.5 volumes of ethanol. After overnight incubation at -20.degree. C., the RNA is recovered by centrifugation.
For the inactivation of nucleases in ribonuclease protection assays, the method presently recommended in the literature involves treating the reaction mixture with proteinase K (a protease enzyme that degrades the nuclease) (Sambrook, et al., 1989). This is typically performed in the presence of the ionic detergent sodium dodecyl sulfate (SDS), followed by phenol/chloroform extraction. After this organic extraction, each reaction mixture in the assay series is transferred to a new microcentrifuge tube. Extraneous nucleic acid is frequently added to the reaction after the organic extraction to act as a carrier to aid in the quantitative precipitation of the RNA. The RNA is then recovered by alcohol precipitation, solubilized in a suitable buffer, and analyzed, usually by electrophoresis on thin polyacrylamide gels. A typical protocol for ribonuclease inactivation and recovery of RNA from a ribonuclease protection assay is given in Molecular Cloning: A Laboratory Manual (Sambook, et al., 1989).
Although the isolation of RNA from biological sources and recovery of RNA from nuclease protection assays are similar in some respects, there are more differences than similarities. For example, the protein concentration in nuclease protection assays is low, whereas during RNA isolation from cells or tissue, protein is present in much higher concentrations. Other contaminants present in the cell or tissue homogenates are polysaccharides, cell membranes, DNA and debris derived from organelles. These contaminants are not present in the in vitro nuclease protection reaction mixture. Thus, procedures for RNA isolation must include steps not only for nuclease inactivation, but also for selective removal of protein and other contaminants. In contrast, the removal of protein and other contaminants from in vitro nuclease protection assays is not required, since the only protein present is the nuclease itself.
Since the experimental basis for nuclease protection assays is the selective hybridization of the mRNA being detected to a specific complementary RNA or DNA probe, reaction conditions are chosen that maximize hybridization. Subsequent to the hybridization, the reaction is adjusted to allow nuclease digestion of unhybridized probe. Therefore, components of the in vitro hybridization and nuclease digestion reactions must be considered when the reaction is finally adjusted for the inactivation of the nucleases and recovery of the hybridized RNA. In contrast, the isolation of RNA from biological sources can be carried out in a reaction mixture that is optimized only for the selective recovery of intact RNA.
Nuclease protection assays require the quantitative recovery of small amounts of labeled RNA (usually radioisotopically labeled), while cellular RNA isolation involves selective recovery of unlabeled RNA from complex mixtures containing relatively high concentrations of RNA. To aid in the quantitative recovery of the small amount of RNA in the nuclease protection reactions, a carrier such as tRNA or yeast RNA is often added to increase the total RNA concentration.
From a practical standpoint, it is important that the procedure for recovery of RNA from in vitro nuclease protection assays be rapid and simple enough to permit the recovery of RNA from multiple samples (on the order of 10-20 samples), since many samples are usually analyzed in parallel. In contrast, while speed and simplicity are obvious advantages in procedures for cellular RNA isolation, they are not critical parameters since the RNA is usually being isolated from only one or a few sources.
By their nature, nuclease protection assays require the application of multiple steps. Innovations that make any of these steps less cumbersome and time consuming will enhance the usefulness of the assay and broaden its appeal. As previously performed, nuclease protection assays require the application of multiple steps which can be cumbersome and time consuming. For example, previous assays require the use of proteinase digestion and organic extraction to remove enzymes prior to RNA precipitation. Organic extraction is a step that involves a significant degree of handling and can result in a loss of desired nucleic acids into the organic phase. There is, in fact, an overall need to improve the efficiency of recovery of nucleic acids from protection assays. Such improvements could go a long way towards simplifying these assays, opening the possibility of automation and making them more readily practiced by less trained laboratory personnel, freeing such individuals to carry out other work.